Death and Resurrection of an Inhibitory Na+ Binding Site in a Human Epithelial Na+ Channel Subunit

Epithelial Na+ channels (ENaC) transport and sense Na+ in a variety of tissues. These channels selectively transport Na+ and are also inhibited by it, an effect called Na+ self-inhibition. ENaCs are heterotrimers comprising α, β, and γ subunits. A fourth ENaC subunit δ, related by gene duplication to α, can replace the α subunit and confer unique functional properties. Among these are Na+ self-inhibition, present in αβγ, but absent in δβγ. We previously identified a putative Na+ binding site centered around mouse αD365. Functional data provided evidence that a homologous site was present in the Xenopus α and δ subunits, and structural data provided evidence for a cation bound at the homologous site in the human α subunit (D338). We hypothesized that 1) the α/δ ancestral subunit had an inhibitory Na+ binding site, 2) that the site was lost on the δ subunit lineage, and 3) that the human δ subunit site could be resurrected. We determined a phylogenetic tree from >800 α and δ subunit sequences, and examined substitution at the position of the key aspartate. Among the α subunits, the key aspartate was omnipresent, absent only in the sloth. Among the δ subunits, the key aspartate was absent in >60 species, including primates and other mammalian orders, and some perching birds. By contrasting nested models of site evolution, we found that site retention was 67-fold more favorable for α than for δ (p=0.02). When we measured non-synonymous to synonymous substitution rate ratios (dN/dS), we found that the α subunit was under purifying selection (p < 0.001) while the δ subunit was not (p = 0.4), providing evidence for a loss of selection pressure after the divergence of the α and δ subunits. Results for other residues at the putative inhibitory Na+ binding site were similar. The treeshrew ( Tupaia belangerie) was the closest primate relative with a putative Na+ self-inhibition site in its δ subunit. We confirmed the site’s functionality by complementary expression with human β and γ subunits in Xenopus oocytes and performing two-electrode voltage clamp experiments. Mutating the homologous aspartate in treeshrew δ significantly reduced Na+ self-inhibition (p < 0.0001). To confer Na+ self-inhibition to human δ, we mutated residues at the putative site to their treeshrew δ equivalents. Restoring the key aspartate in human δ (dP314D) resulted in discernable Na+ self-inhibition (p=0.003), and treeshrew-ifying 2 additional sites (G136M and L322E) conferred Na+ self-inhibition in human δ like that measured in human α. We then examined ENaC subunit expression in tissues from several species. In contrast to Xenopus laevis, we did not detect δ subunit transcripts in kidneys from the treeshrew, chicken, or opossum. Our study provides evidence that the site centered around human aD338 is a bona fide allosteric Na+ binding site, and that selection pressure maintained this site in the α subunit but not the δ subunit, possibly due to differential renal expression. DK125439. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.